Bruno Detry

Rise and Survival of Bovine Herpesvirus 1 Recombinants after Primary Infection and Reactivation from Latency

ABSTRACT Recombination is thought to be an important source of genetic variation in herpesviruses. Several studies, performed in vitro or in vivo, detected recombinant viruses after the coinoculation of two distinguishable strains of the same herpesvirus species. However, none of these studies investigated the evolution of the relative proportions of parental versus recombinant progeny populations after coinoculation of the natural host, both during the excretion and the reexcretion period. In the present study, we address this by studying the infection of cattle with bovine herpesvirus 1 (BoHV-1). The recombination of two BoHV-1 mutants lacking either glycoprotein C (gC−/gE+) or E (gC+/gE−) was investigated after inoculation of cattle by the natural route of infection. The results demonstrated that (i) recombination is a frequent event in vivo since recombinants (gC+/gE+ and gC−/gE−) were detected in all coinoculated calves, (ii) relative proportions of progeny populations evolved during the excretion period toward a situation where two populations (gC+/gE+ and gC−/gE+) predominated without fully outcompeting the presence of the two other detected populations (gC+/gE− and gC−/gE−), and (iii) after reactivation from latency, no gC+/gE− and gC−/gE− progeny viruses were detected, although gC+/gE− mutants, when inoculated alone, were detected after reactivation treatment. In view of these data, the importance of gE in the biology of BoHV-1 infection and the role of recombination in herpesvirus evolution are discussed.

Investigation of the Susceptibility of Human Cell Lines to Bovine Herpesvirus 4 Infection: Demonstration that Human Cells Can Support a Nonpermissive Persistent Infection Which Protects Them against Tumor Necrosis Factor Alpha-Induced Apoptosis

ABSTRACT Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus that has a worldwide distribution in the population of cattle. Many factors make human contamination by BoHV-4 likely to occur. In this study, we performed in vitro experiments to assess the risk and the consequences of human infection by BoHV-4. First, by using a recombinant BoHV-4 strain expressing enhanced green fluorescent protein under the control of the human cytomegalovirus immediate-early gene promoter, we tested 21 human cell lines for their sensitivity and their permissiveness to BoHV-4 infection. These experiments revealed that human cell lines from lymphoid and myeloid origins were resistant to infection, whereas epithelial cells, carcinoma cells, or adenocarcinoma cells isolated from various organs were sensitive but poorly permissive to BoHV-4 infection. Second, by using the HeLa cell line as a model of human cells sensitive but not permissive to BoHV-4 infection, we investigated the resistance of infected cells to apoptosis and the persistence of the infection through cellular divisions. The results obtained can be summarized as follows. (i) BoHV-4 nonpermissive infection of HeLa cells protects them against tumor necrosis factor alpha-induced apoptosis. (ii) BoHV-4 infection of HeLa cells persists in cell culture; however, the percentage of infected cells decreases with time due to erratic transmission of the viral genome through cell division. (iii) BoHV-4 infection has no effect on the rate of HeLa cell division. Altogether, these data suggest that BoHV-4 could infect humans. This study also stresses the importance of considering the insidious effects of nonpermissive infection when the biosafety of animal gammaherpesviruses for humans is being considered.

Improved Antigenic Methods for Differential Diagnosis of Bovine, Caprine, and Cervine Alphaherpesviruses Related to Bovine Herpesvirus 1

ABSTRACT The control of infectious bovine rhinotracheitis induced by bovine herpesvirus 1 (BoHV-1) requires sensitive and specific diagnostic assays. As BoHV-1 is antigenically and genetically related to four other alphaherpesviruses of ruminants—namely, BoHV-5, caprine herpesvirus 1 (CpHV-1), cervine herpesvirus 1 (CvHV-1) and CvHV-2—diagnostic tests able to discriminate BoHV-1 from these related viruses are needed to avoid misdiagnosis, especially because some of these viruses are able to cross the species barrier. In this study, murine monoclonal antibodies (MAbs) specific for BoHV-1, BoHV-5, CpHV-1, CvHV-1, and CvHV-2 were produced with the aim of setting up an immunofluorescence assay able to discriminate between these related herpesviruses. Produced MAbs were selected for their viral specificity by enzyme-linked immunosorbent assay and indirect immunofluorescence staining of virus-infected cells. Radioimmunoprecipitation characterization of the selected MAbs revealed that four of them are directed against glycoprotein C (gC) and one of them is directed against gD of these related viruses. The obtained results demonstrate that the antibodies produced allow an unambiguous discrimination of each of the four alphaherpesviruses related to BoHV-1.

The structures of bovine herpesvirus 1 virion and concatemeric DNA: implications for cleavage and packaging of herpesvirus genomes

Herpesvirus genomes are often characterized by the presence of direct and inverted repeats that delineate their grouping into six structural classes. Class D genomes consist of a long (L) segment and a short (S) segment. The latter is flanked by large inverted repeats. DNA replication produces concatemers of head-to-tail linked genomes that are cleaved into unit genomes during the process of packaging DNA into capsids. Packaged class D genomes are an equimolar mixture of two isomers in which S is in either of two orientations, presumably a consequence of homologous recombination between the inverted repeats. The L segment remains predominantly fixed in a prototype (P) orientation; however, low levels of genomes having inverted L (I(L)) segments have been reported for some class D herpesviruses. Inefficient formation of class D I(L) genomes has been attributed to infrequent L segment inversion, but recent detection of frequent inverted L segments in equine herpesvirus 1 concatemers [Virology 229 (1997) 415-420] suggests that the defect may be at the level of cleavage and packaging rather than inversion. In this study, the structures of virion and concatemeric DNA of another class D herpesvirus, bovine herpesvirus 1, were determined. Virion DNA contained low levels of I(L) genomes, whereas concatemeric DNA contained significant amounts of L segments in both P and I(L) orientations. However, concatemeric termini exhibited a preponderance of L termini derived from P isomers which was comparable to the preponderance of P genomes found in virion DNA. Thus, the defect in formation of I(L) genomes appears to lie at the level of concatemer cleavage. These results have important implications for the mechanisms by which herpesvirus DNA cleavage and packaging occur.

Induce triploidy by heat shock in Eurasian perch, Perca fluviatilis

In Eurasian perch (Perca fluviatilis), females grow significantly faster than males. Moreover, gonadal development has a significant negative impact on somatic growth and fillet yield. In order to induce sterility, triploidy induction was attempted by subjecting fertilised eggs to heat shocks. Different combinations of temperature (28, 30, 34, 35 and 36 °C), duration (2, 5, 10 and 25 min) and time of shock initiation (TI = 3, 5 and 7 min post-fertilisation) were tested. Flow cytometry analysis was used to assess ploidy level of control and heat-shocked larvae. Low intensity (28–30 °C) and long duration (10 and 25 min) shocks lead to significantly higher survival (44 ± 26%) and triploidisation (71 ± 26%) rates than high intensity (34–36 °C) and short duration (2 and 5 min) shocks (17 ± 19% and 21 ± 26%, respectively). The most effective conditions for efficient triploidy induction were low intensity shock of 30 °C, applied 5 min post-fertilisation for 25 min. This treatment led to the production of all-triploid populations (100%) with up to 43% survival rate.